Blood coagulation is a process consisting of a complex interaction of various blood components which eventually gives rise to a fibrin network, or clot. Degradation of the fibrin network can be accomplished by activation of the zymogen plasminogen into plasmin, a serine protease which acts directly to degrade the fibrin network and thereby regulate the coagulation process. Conversion of plasminogen into plasmin is normally catalyzed in vivo by tissue-type plasminogen activator (t-PA), a fibrin-specific serine protease which is believed to be the physiological vascular activator of plasminogen. Urokinase-type plasminogen activator (u-PA) is another member of the class of plasminogen activators characterized as serine proteases. t-PA and u-PA are functionally and immunologically distinguishable.
t-PA normally circulates as a single polypeptide chain of M.sub.r .perspectiveto.72,000 daltons which is converted to a two-chain form by cleavage of a peptide bond between amino acids 275 (Arg) and 276 (Ile). This cleavage is catayzed by trypsin or plasmin, and is accompanied by an increase in activity as measured using synthetic substrates, and by an increase in fibrinolytic activity. Fibrinolytic activity of t-PA is enhanced upon binding to fibrin, due in part to the high concentrations of plasminogen and t-PA present on the surface of a fibrin clot. Cleavage to the two-chain form may also be associated with rapid clearance of t-PA from the bloodstream, but conflicting reports on this have been published (see Wallen et al., Eur. J. Biochem. 132: 681-686, 1983).
A two-dimensional model of the potential precursor t-PA protein has been established (Ny et al., Proc. Natl. Acad. Sci. USA 81: 5355-5359, 1984). From this model, it was determined that the heavy chain contains two triple disulfide structures known as "kringles." These kringle structures also occur in prothrombin, plasminogen and urokinase, and are believed to be important for binding to fibrin (Ny et al., ibid.). The second kringle (K.sub.2) of t-PA is believed to have a higher affinity for fibrin than the first kringle (K.sub.1) (Ichinose, Takio and Fujikawa, J. Clin. Invest. 78: 163-169, 1986). The heavy chain of t-PA (two variants of M.sub.r 40,000 and 37,000) is derived from the amino-terminus, while the light chain (M.sub.r 33,000) is derived from the carboxy-terminal end of the t-PA molecule.
The heavy chain of t-PA also contains a "finger" domain that is homologous to the finger domains of fibronectin. Fibronectin has been implicated in a variety of biological activities, including fibrin binding, and the fibrin binding activity has been correlated to four or five of the nine finger domains possessed by fibronectin. The heavy chain of t-PA also contains a growth factor-like domain.
The light chain of t-PA contains the active site for serine protease activity, which is highly homologous to the active sites of other serine proteases.
Native t-PA additionally comprises a pre-region followed downstream by a pro-region, which are collectively referred to as the "pre-pro" region. The pre-region contains a signal peptide which is important for secretion of t-PA by vascular endothelial cells (Ny et al., ibid.). The pre sequence is believed responsible for secretion of t-PA into the lumen of the endoplasmic reticulum, a necessary step in extracellular secretion. The pro sequence is believed to be cleaved from the t-PA molecule following transport from the endoplasmic reticulum to the Golgi apparatus.
Urokinase exhibits structural features similar to those of t-PA. The precursor form of the molecule has the structure pre-pro-growth factor-kringle-serine protease. The zymogen pro-urokinase is activated upon cleavage at an activation site between the kringle and serine protease domains by plasmin, trypsin, or plasma kallikrein (Ichinose et al., J. Biol. Chem. 261: 3486-3489, 1986). Urokinase also contains a thrombin cleavage site adjacent to the activation site. Cleavage by thrombin results in inactivation of the protein (Ichinose et al., ibid.).
The structure and activation of plasminogen are reviewed by Collen (Thromb. Haemostasis 43: 77-89, 1980). Briefly, plasminogen is a single-chain glycoprotein of M.sub.r .perspectiveto.90,000 (designated "Glu-plasminogen") comprising five kringle structures and a carboxyl-terminal serine protease domain. Activation of plasminogen to plasmin is a multistep process involving the removal of an aminoterminal peptide (conversion to Lys-plasminogen) and cleavage at the activation site to produce the active, two-chain protein. Glu-plasminogen may also be cleaved at the activation site, followed by removal of the amino-terminal peptide.
Urokinase has a low affinity for fibrin and activates plasminogen throughout the body. While t-PA is more specific for cleavage of plasminogen in the presence of fibrin (i.e., at the site of a clot), therapeutic doses of t-PA sufficient to lyse coronary thrombi are far larger than normal physiological levels. Further, doses of this size may also activate plasminogen throughout the body, leading to systemic degradation of fibrinogen (Sherry, New Eng. J. Med. 313: 1014-1017, 1985), which results in dangerous bleeding episodes. The systemic activity of t-PA may be due to activation by a low level of free plasmin in the circulation or to proteolytic activity of the two-chain form of t-PA used in the clinical studies.
These side effects of t-PA have been recognized, and several approaches have been employed in an attempt to create an improved form of t-PA. For example, Heyneker and Vehar (GB 2,173,804) disclose modified t-PA molecules alleged to have increased specificity. In addition, Rosa and Rosa (International Patent Application WO 86/01538) modified the sequence around the activation site of t-PA with the aim of increasing the half-life.
Although these attempts have met with some degree of success, within clinical applications, it would be advantageous to employ fibrinolytic agents possessing a higher specificity for catalytic activity at the clot site than is observed for the naturally-occurring plasminogen activators. The present invention fulfills the need for a fibrinolytic agent which combines the clinical efficacy of t-PA with minimal undesirable side effects by providing novel, highly specific fibrinolytic agents which may be produced in relatively large quantities. Through the use of recombinant DNA technology, a consistent and homogeneous source of these fibrinolytic agents is provided. These fibrinolytic agents can be utilized to lyse existing clots in heart attack and stroke victims, and in other patients where the need to lyse clots or suppress the formation of fibrin matrices is therapeutically desirable.